title: Location of subunit d in the peripheral stalk of the ATP synthase from Saccharomyces cerevisiae.
abstract: ATP synthase from Saccharomyces cerevisiae is an approximately 600 kDa membrane protein complex. The enzyme couples the proton motive force across the mitochondrial inner membrane to the synthesis of ATP from ADP and inorganic phosphate. The peripheral stalk subcomplex acts as a stator, preventing the rotation of the soluble F 1 region relative to the membrane-bound F O region during ATP synthesis. Component subunits of the peripheral stalk are Atp5p (OSCP), Atp4p (subunit b), Atp7p (subunit d), and Atp14p (subunit h). X-ray crystallography has defined the structure of a large fragment of the bovine peripheral stalk, including 75% of subunit d (residues 3-123). Docking the peripheral stalk structure into a cryo-EM map of intact yeast ATP synthase showed that residue 123 of subunit d lies close to the bottom edge of F 1. The 37 missing C-terminal residues are predicted to either fold back toward the apex of F 1 or extend toward the membrane. To locate the C terminus of subunit d within the peripheral stalk of ATP synthase from S. cerevisiae, a biotinylation signal was fused to the protein. The biotin acceptor domain became biotinylated in vivo and was subsequently labeled with avidin in vitro. Electron microscopy of the avidin-labeled complex showed the label tethered close to the membrane surface. We propose that the C-terminal region of subunit d spans the gap from F 1 to F O, reinforcing this section of the peripheral stalk.
authors.affiliation: Molecular Structure and Function Program, The Hospital for Sick Children Research Institute.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Bueler
authors.forename: John L
authors.initials: JL
authors.lastname: Rubinstein
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid
chemical_list: D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D025261:Mitochondrial Proton-Translocating ATPases